Novel methods of treating a neurodegenerative disease in a mammal in need thereof

ABSTRACT

The present invention provides a method of treating or ameliorating a neurodegenerative disease in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a neurodegenerative disease drug, wherein the drug is a substrate of an ABC transporter inhibitor, wherein the mammal is further administered a therapeutically effective amount of an ABC transporter inhibitor, whereby the neurodegenerative disease is treated in the mammal. In certain embodiments, the neurodegenerative disease comprises at least one selected from the group consisting of spinal cord injury, Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, prion disease, amyotrophic lateral sclerosis, a tauopathy, and chronic traumatic encephalopathy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 61/913,738, filed Dec. 9, 2014, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberAL100153 awarded by the U.S. Department of Defense and grant number RO1NS74886 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease,is a debilitating disease with varied etiology. ALS is characterized byrapidly progressive weakness, muscle atrophy and fasciculations, musclespasticity, difficulty speaking (i.e., dysarthria), difficultyswallowing (i.e., dysphagia) and difficulty breathing (i.e., dyspnea).Worldwide, ALS kills about 100,000 yearly, with 125,000 new ALSdiagnoses yearly.

ALS causes muscle weakness and atrophy throughout the body due to thedegeneration of the upper and lower motor neurons. Individuals affectedby the disorder may ultimately lose the ability to initiate and controlall voluntary movement, although bladder and bowel sphincters and themuscles responsible for eye movement are usually spared until theterminal stages of the disease. Most patients do not lose cognitivefunction, but about 5% develop frontotemporal dementia.

The precise cause of ALS is still unknown, but mutations in the genethat produces the Cu/Zn superoxide dismutase (SOD1) enzyme are presentin approximately 20% of familial ALS cases. SOD1 is a powerfulantioxidant that protects the body from damage caused by the toxic freeradical superoxide, which is generated in the mitochondria. To date,over 110 different mutations in SOD1 have been linked with ALS. Failureof defenses against oxidative stress upregulates programmed cell death(apoptosis). An accumulation of free radicals due to faulty SOD1functioning may thus lead to motor neuron degeneration. However, it isalso possible that mutant SOD1 induces toxicity by a gain of function.For example, in some mutant SOD1 mice, mutant SOD1 aggregates (misfoldedprotein deposits) were found only in diseased tissues, and greateramounts were detected during motor neuron degeneration. This observationis consistent with a model where mutant SOD1 aggregates help disruptcellular functions by damaging mitochondria, proteasomes or proteinfolding chaperones. In humans, SOD1 mutations cause only 2% of total ALScases, and the etiological mechanisms may be distinct from thoseresponsible for the sporadic form of the disease. For non-familial cases(around 90% of ALS cases), the disease may be triggered by head trauma,military service, or participation in contact sports.

Riluzole (RILUTEK®) is the only drug currently approved to treat ALS,but its therapeutic success is limited. Riluzole slows down diseaseprogression by about 3-9 months, prolongs survival by several months,and delays the need for ventilation support. However, the drug does notreverse the damage already done to motor neurons, and may cause liverdamage in about 10% of the patients.

In one aspect, development of effective ALS treatments may be hamperedby a combination of low drug bioavailability and disease-drivenpharmacoresistance, which limits CNS drug penetration and ultimatelycompromises drug efficacy.

Pharmacoresistance is mediated for example by the ATP-binding cassette(“ABC”) drug efflux transporters. ABC transporters are transmembraneproteins that utilize the energy of adenosine triphosphate (ATP)hydrolysis to carry out biological processes, including translocation ofsubstrates (e.g., metabolites, lipids, sterols and drugs) across extra-and intracellular membranes. ABC transporters play a crucial role in thedevelopment of multidrug resistance (MDR), whereby patients eventuallydevelop resistance not only to the drug they are taking but also toadditional types of drugs to which they had not been exposed. MDR islinked by the increased cellular excretion of the drug by ABCtransporters. ABC transporter families include ABCB1 (also known asP-glycoprotein, P-gp or MDR1), which transports organic cationiccompounds or neutral compounds; MRP, which transports organic anioniccompounds; and ABCG2 (also known as breast cancer resistance protein orBCRP), which confers resistance to Topoisomerase I or II inhibitors suchas topotecan, irinotecan, and doxorubicin. ABC transporters arepredominantly located in the lumen of endothelial cells of theblood-brain barrier, where they limit the entry of neurotoxins into theCNS. Unfortunately, clinical trials in cancer patients to which ABCtransporter inhibitors were co-administered, with the objective ofincreasing the anticancer drug efficacy, had ultimately disappointingoutcomes (Sharom, 2008, Pharmacogenomics 9(1):105-127; Szakacs et al.,2006, Nat. Rev. Drug Discov. 5:219-234).

There is a need in the art for novel methods of treatingneurodegenerative diseases in a mammal in need thereof. The presentinvention fulfills this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a pharmaceutical composition comprising aneurodegenerative disease drug and an ABC transporter inhibitor, whereinthe drug is a substrate of an ABC transporter.

The inventor further includes a method of treating or ameliorating aneurodegenerative disease in a mammal in need thereof, the methodcomprising administering to the mammal a therapeutically effectiveamount of a neurodegenerative disease drug, wherein the drug is asubstrate of an ABC transporter, wherein the mammal is furtheradministered a therapeutically effective amount of an ABC transporterinhibitor, whereby the neurodegenerative disease is treated orameliorated in the mammal.

In certain embodiments, the neurodegenerative disease comprises at leastone selected from the group consisting of spinal cord injury,Alzheimer's disease, Parkinson's disease, Huntington's disease, priondisease, amyotrophic lateral sclerosis (ALS), a tauopathy, and chronictraumatic encephalopathy. In other embodiments, the disease comprisesALS.

In certain embodiments, the ABC transporter comprises at least oneselected from the group consisting of P-gp and BRCP.

In certain embodiments, the drug comprises at least one selected fromthe group consisting of ceftriaxone; celecoxib; ciliary neurotrophicfactor; cobalamin; coenzyme Q; gabapentin; HGF; IGF-I; minocycline;N-acetylcysteine; NDGA; pentoxifylline; riluzole; thalidomide;topiramate; valproic acid; VEGF; vitamin E; zVAD-fmk; a salt or solvatethereof, and any mixtures thereof.

In certain embodiments, the inhibitor comprises at least one selectedfrom the group consisting of elacridar; tariquidar; zosuquidar; ONT-093;laniquidar; a salt or solvate thereof, and mixtures thereof.

In certain embodiments, the composition is formulated for inhalational,oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary,intranasal, buccal, sublingual, ophthalmic, intrathecal, intravenousand/or intragastrical administration. In other embodiments, the drug isadministered to the mammal by at least one route selected from the groupconsisting of inhalational, oral, rectal, vaginal, parenteral, topical,transdermal, pulmonary, intranasal, buccal, sublingual, ophthalmic,intrathecal, intravenous and intragastrical. In yet other embodiments,at least one selected from the group consisting of the drug andinhibitor is part of a pharmaceutical composition. In yet otherembodiments, the pharmaceutical composition comprises anextended-release formulation. In yet other embodiments, the drug and theinhibitor are co-administered to the mammal. In yet other embodiments,the drug and the inhibitor are coformulated.

In certain embodiments, administration to the mammal takes place oncethe mammal develops any symptom of the neurodegenerative disease. Inother embodiments, the mammal that is administered the drug and theinhibitor has a higher spinal cord concentration of the drug than amammal that is administered the drug only. In yet other embodiments, themammal that is administered the drug and the inhibitor has a highercompound muscle action potential peak amplitude than a mammal that isadministered the drug only. In yet other embodiments, the mammal that isadministered the drug and the inhibitor has improved survival ascompared to a mammal that is administered the drug only. In yet otherembodiments, the mammal that is administered the drug and the inhibitorhas delayed disease progression as compared to a mammal that isadministered the drug only. In yet other embodiments, the mammalexperiences no significant hepatotoxicity when administered the drug andthe inhibitor.

In certain embodiments, the mammal is a rodent or a primate. In otherembodiments, the primate is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings. FIGS. 1A-1C illustrate the findingthat ALS patients have tissue-specific increases in P-gp proteinexpression. Protein expression of P-gp in ALS patients compared tocontrols and levels of P-gp in diseased versus non-diseased tissue areaswere examined. FIG. 1A: Densitometric analysis of P-gp proteinexpression in lumbar spinal cord showed an increase (1.2- to 1.5-fold,depending on the patient) in sporadic (sALS) and familial (fALS) ALScompared to non-neuromuscular controls and a patient with Friedreich'sAtaxia, a different neurological, spinal neuron disease. FIG. 1B:Immunohistochemistry of lumbar spinal cord sections shows thecolocalization of P-gp with the endothelial cell marker, von WillebrandFactor. P-gp expression was higher in endothelial cells of ALS patientsin the ventral horn of lumbar spinal cord compared to controls (scalebar=20 μm). FIG. 1C: Levels of P-gp expression in the lumbar spinal cordand the hippocampus, an unaffected brain region, of the same ALS patientwere compared. P-gp expression increases are tissue specific with higherlevels of P-gp expression in the spinal cord of the ALS patient comparedto the unaffected area (hippocampus) (scale bar=20 μm). Thus, in ALSpatients there was a tissue-specific increase in P-gp proteinexpression, which was selective to endothelial cells at the level of theBSCB.

FIGS. 2A-2E illustrate the finding that P-gp substrate disposition isaltered in SOD1-G93A mice compared to wild-type (WT), and geneticinhibition of P-gp increases the effectiveness of riluzole in SOD1-G93Amice. A specific P-gp substrate, LD800, was injected intraperitoneallyinto WT, P-gp knockout (P-gp^(−/−)), and symptomatic SOD1-G93A mice.Symptomatic SOD1-G93A mice had decreased accumulation of LD800 into thespinal cord compared to WT and P-gp^(−/−) mice (P=0.021 and P<0.001,respectively; FIGS. 2A and 2B). Riluzole is a P-gp substrate and hasincreased spinal cord (FIG. 2C) and total CNS (FIG. 2D) penetration inP-gp knockout mice. WT and P-gp^(−/−) mice were acutely treated withintraperitoneal injections of riluzole, and the concentrations ofriluzole in the plasma and spinal cord tissue were determined via massspectrometry. FIG. 2C: The percent of riluzole accumulation, normalizedto riluzole plasma concentrations, in the spinal cords of P-gp^(−/−)mice was significantly higher than the amount of riluzole in WT mice(20.9±4.3% and 8.4±2.2%, respectively; P=0.028). FIG. 2D: Accumulationof riluzole in the total CNS (brain and spinal cord) was alsosignificantly higher in P-gp^(−/−) compared to WT mice (31.9±4.7% and15.6±2.0%, respectively; P=0.010). FIG. 2E: As compared to untreatedP-gp^(−/−)::SOD1-G93A mice and SOD1-G93A riluzole-treated mice,P-gp^(−/−)::SOD1-G93A riluzole-treated mice had a trend toward increasedsurvival (165.2±2.89, 162.0±2.51, and 176.0±4.2, respectively; n=5-6).

FIGS. 3A-3E illustrate that chronic elacridar treatment alone does notalter survival or P-gp expression levels, but does increase riluzoleaccumulation in SOD1-G93A mouse spinal cord. FIG. 3A: Chronic treatmentof elacridar, beginning at disease onset, was safe and did not alterdisease progression in the SOD1-G93A ALS mice (Log-rank Mantel-Cox,χ²=0.046, P=0.830). FIG. 3B: P-gp expression levels in riluzole andriluzole+elacridar-treated mice were not altered. FIG. 3C: Penetrationof riluzole in the spinal cord of symptomatic 140-day-old miceco-treated with elacridar was measured by mass spectrometry compared toriluzole-only-treated mice. Significantly higher levels of riluzole weredetected in riluzole+elacridar-treated mice compared toriluzole+placebo-treated aged-matched 140-day-old ALS mice (295±70.9%;P=0.016). FIG. 3D: Periodic acid Schiff staining of liver sections fromriluzole+placebo-treated mice and riluzole+elacridar-treated mice (scalebar=50 μm). Chronic treatment with riluzole and elacridar did not causeovert toxicity to the liver of SOD1-G93A mice compared to riluzoletreatment alone. FIG. 3E: SOD1-G93A study mice had the same levels ofthe human transgene as quantified by qRT-PCR of DNA isolated from mousetail. Mice displaying higher or lower copy numbers were excluded.

FIGS. 4A-4G illustrate that cotreatment with riluzole and elacridarincreases survival, NMJ function, behavior, and motor neuron countscompared to riluzole treatment alone. FIG. 4A: Cotreatment ofriluzole+elacridar (165±2.1 days) significantly extended survivalcompared to control+placebo (156.7±2.8 days) and riluzole+placebo(159.7±1.9 days) groups (Log-rank Mantel-Cox, χ²=7.292, P=0.026; Logranktest for trend, χ²=6.615, P=0.010; Gehan-Breslow-Wilcoxon, χ²=8.226,P=0.016). FIGS. 4B-4C: Compound muscle action potentials (CMAPs) hadhigher peak amplitudes in 140 day riluzole+elacridar-treated micecompared to age-matched riluzole+placebo-treated mice (7.48±0.67 mV and5.00±0.67 mV, respectively; P=0.017). FIG. 4D: Riluzole+elacridartreatment sustained higher CMAP values throughout treatment compared toriluzole+placebo treatment. FIG. 4E: In the riluzole+elacridar-treatedmice, the slope of hind limb strength decline was significantlydecreased compared to the riluzole+placebo group (P<0.001). FIGS. 4F-4G:Quantification (neurons larger than 400 μm) and stained lumbar spinalcord tissue sections of motor neurons in the ventral horn of SOD1-G93Amice. Food intake did not differ significantly between the any of thegroups.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected discovery thatco-administration of an ABC transporter inhibitor increases thebioavailability and efficacy of a neurodegenerative disease drug in amammal, wherein the drug is itself a substrate of a ABC transporter. Incertain embodiments, the co-administration of the two compounds causesno significant hepatotoxicity in the mammal.

In one aspect, the invention includes a method of treating aneurodegenerative disease in a mammal in need thereof. In certainembodiments, the method comprises administering to the mammal atherapeutically effective amount of a neurodegenerative disease drug,wherein the mammal is further administered at least one ABC transporterinhibitor.

Research has identified several pathogenic mechanisms underlying ALS,but unfortunately clinical trials designed to interfere with these toxicmechanisms have not been successful. One possible explanation for thesefailures may be the development of pharmacoresistance, which compromisesthe overall bioavailability of the drugs tested. To this daypharmacoresistance has not been thoroughly studied in the context ofALS.

As reported herein, the inventors have unexpectedly improved thebioavailability and efficacy of an ALS drug by blocking acquiredpharmacoresistance. In a non-limiting example, a combination therapy wassuccessfully used in the SOD1-G93A ALS mouse model. Specifically, thecombination therapy comprised an ABC transporter inhibitor, elacridar,which blocks P-gp and BCRP (two transporters that are involved in ALSpharmacoresistance), and riluzole, the only FDA-approved drug to treatALS. By administering elacridar in combination with riluzole in amammal, riluzole concentrations were increased in the central nervoussystem, motor performance was enhanced, and the lifespan of ALS mice wasincreased as compared to untreated mice or mice treated with riluzoleonly. These studies indicate that riluzole efficacy may be improved byblocking pharmacoresistance in ALS patients.

In one aspect, the present invention is applicable to any ALS drug,currently known or developed anytime in the future, whose activity isdecreased because of pharmacoresistance. In another aspect, the presentinvention is applicable to any neurodegenerative disease drug whichactivity is decreased because of pharmacoresistance. Restricted drugpenetration into the central nervous system is a common challenge intreating neurodegenerative disorders, and combination of theneurodegenerative disease drug with an ABC transporter inhibitor hasbeen discovered herein to improve the bioavailability and efficacy of adrug so restricted. Alterations in ABC transporter expression have beenidentified, in non-limiting examples, in spinal cord injury, Alzheimer'sdisease and Parkinson's disease.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section. Unless defined otherwise, all technical andscientific terms used herein generally have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Generally, the nomenclature used herein and the laboratoryprocedures in cell culture, molecular genetics, organic chemistry, andpharmacology are those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

As used herein, the term “about” is understood by persons of ordinaryskill in the art and varies to some extent on the context in which it isused. As used herein when referring to a measurable value such as anamount, a temporal duration, and the like, the term “about” is meant toencompass variations of±20%,±10%,±5%,±1%, or±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein, the term “ALS” or “Lou Gehrig's disease” may be usedinterchangeably to refer to amyotrophic lateral sclerosis.

As used herein, the term “BCRP” refers to breast cancer resistanceprotein. As used herein, the term “ceftriaxone” refers to(6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino}-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)sulfanyl]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid, or a salt or solvate thereof.

As used herein, the term “celecoxib” refers to4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide,or a salt or solvate thereof.

As used herein, the term “CNS” refers to central nervous system.

As used herein, the term “elacridar” refers toN-[4-[2-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)ethyl]phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridinecarboxamide, or a salt or solvate thereof.

As used herein, the term “gabapentin” refers to2-[1-(aminomethyl)cyclohexyl] acetic acid, or a salt or solvate thereof.

As used herein, the term “HGF” refers to hepatocyte growth factor.

As used herein, the term “IGF-I” refers to insulin-like growth factor 1.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionthat may be used to communicate the usefulness of the compounds and/orcompositions of the invention. In some instances, the instructionalmaterial may be part of a kit useful for effecting alleviating ortreating the various diseases or disorders recited herein. Optionally,or alternately, the instructional material may describe one or moremethods of alleviating the diseases or disorders in a cell or a tissueof a mammal. The instructional material of the kit may, for example, beaffixed to a container that contains the compounds and/or compositionsof the invention or be shipped together with a container that containsthe compounds and/or compositions. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the recipient uses the instructional material and the compoundand/or composition cooperatively. For example, the instructionalmaterial is for use of a kit; instructions for use of the compoundand/or composition; or instructions for use of a formulation of thecompound and/or composition.

As used herein, the term “laniquidarmethyl” refers to11-(1-{2-[4-(quinolin-2-ylmethoxy)phenyl]ethyl}piperidin-4-ylidene)-6,11-dihydro-5H-imidazo[2,1-b][3]benzazepine-3-carboxylate,or a salt or solvate thereof.

As used herein, the term “minocycline” refers to(2E,4S,4aR,5aS,12aR)-2-(aminohydroxymethylidene)-4,7-bis(dimethylamino)-10,11,12a-trihydroxy-4a,5,5a,6-tetrahydro-4H-tetracene-1,3,12-trione,or a salt or solvate thereof. As used herein, the term “NDGA” refers to4,4′-(2,3-dimethylbutane-1,4-diyl) dibenzene-1,2-diol, or a salt orsolvate thereof.

As used herein, a “neurodegenerative disease drug” refers to a compound(such as but not limited to a small molecule, nucleic acid, peptide,protein or antibody) that is useful in treating a neurodegenerativedisease, a symptom of a neurodegenerative disease or the potential todevelop a neurodegenerative disease. In certain embodiments,administration of the neurodegenerative drug has the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe neurodegenerative disease, the symptoms of the neurodegenerativedisease, or the potential to develop the neurodegenerative disease.

As used herein, the term “ONT-093” refers to(E)-4,4′-(2-(4-(3-ethoxyprop-1-en-1-yl)phenyl)-1H-imidazole-4,5-diyl)bis(N-isopropylaniline),or a salt or solvate thereof.

As used herein, the term “patient” or “individual” or “subject” refersto a human or a non-human mammal. Non-human mammals include, forexample, livestock and pets, such as ovine, bovine, porcine, canine,feline and murine mammals. In certain embodiments, the patient,individual or subject is human.

As used herein, the term “pentoxifylline” refers to3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1H-purine-2,6-dione, or a saltor solvate thereof.

As used herein, the terms “pharmaceutically effective amount” and“effective amount” and “therapeutically effective amount” referinterchangeably to a nontoxic but sufficient amount of an agent toprovide the desired biological result. That result may be reductionand/or alleviation of the signs, symptoms, or causes of a disease, orany other desired alteration of a biological system. An appropriatetherapeutic amount in any individual case may be determined by one ofordinary skill in the art using routine experimentation.

As used herein, the term “P-gp” or “Pgp” refers to P-glycoprotein.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids and bases, including inorganic acids,inorganic bases, organic acids, inorganic bases, solvates, hydrates, andclathrates thereof. Suitable pharmaceutically acceptable acid additionsalts may be prepared from an inorganic acid or from an organic acid.Examples of inorganic acids include sulfate, hydrogen sulfate,hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, andphosphoric acids (including hydrogen phosphate and dihydrogenphosphate). Appropriate organic acids may be selected from aliphatic,cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic andsulfonic classes of organic acids, examples of which include formic,acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic,phenylacetic, mandelic, embonic (pamoic), methanesulfonic,ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid. Suitable pharmaceutically acceptablebase addition salts of compounds of the invention include, for example,ammonium and metallic salts including alkali metal, alkaline earth metaland transition metal salts such as, for example, calcium, magnesium,potassium, sodium and zinc salts. Pharmaceutically acceptable baseaddition salts also include organic salts made from basic amines suchas, for example, N,N′-dibenzylethylene-diamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. All of these salts may be prepared from the correspondingcompound by reacting, for example, the appropriate acid or base with thecompound.

As used herein, the term “pharmaceutical composition” or “composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient.Multiple techniques of administering a compound exist in the artincluding, but not limited to, intravenous, oral, aerosol, inhalational,rectal, vaginal, transdermal, intranasal, buccal, sublingual,parenteral, intrathecal, intragastrical, ophthalmic, pulmonary andtopical administration.

As used herein, the term “polypeptide” refers to a polymer composed ofamino acid residues, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof linked via peptidebonds. Synthetic polypeptides may be synthesized, for example, using anautomated polypeptide synthesizer. As used herein, the term “protein”typically refers to large polypeptides. As used herein, the term“peptide” typically refers to short polypeptides. Conventional notationis used herein to represent polypeptide sequences: the left-hand end ofa polypeptide sequence is the amino-terminus, and the right-hand end ofa polypeptide sequence is the carboxyl-terminus.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

As used herein, the term “riluzole” refers to6-(trifluoromethoxy)benzothiazol-2-amine, or a salt or solvate thereof.

As used herein, the term “solvate” refers to a complex with one or moresolvent molecules, which may comprise water, methanol, ethanol,1-propanol, 2-propanol, DMSO, DMF, ethyl ether, acetone, and/or MTBE,and the like.

As used herein, the term “substrate” as relating to a drug effluxtransporter refers to a compound (such as a small molecule compound,peptide or protein) that is transported across an extra- orintracellular membrane by the drug efflux transporter.

As used herein, the term “thalidomide” refers to(RS)-2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione, or a saltor solvate thereof.

As used herein, the term “tariquidar” refers toN-[2-[[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide,or a salt or solvate thereof.

As used herein, the term “topiramate” refers to 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate, or a salt or solvatethereof.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compounduseful within the invention (alone or in combination with anotherpharmaceutical agent), to a patient, or application or administration ofa therapeutic agent to an isolated tissue or cell line from a patient(e.g., for diagnosis or ex vivo applications), who has a disease ordisorder, a symptom of a disease or disorder or the potential to developa disease or disorder, with the purpose to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve or affect the disease ordisorder, the symptoms of the disease or disorder, or the potential todevelop the disease or disorder. Such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics.

As used herein, the term “valproic acid” refers to 2-propylpentanoicacid, or a salt or solvate thereof.

As used herein, the term “VEGF” refers to vascular endothelial growthfactor.

As used herein, the term “zVAD-fmk” refers toN-[(phenylmethoxy)carbonyl]-L-valyl-N-[(1S)-3-fluoro-1-(2-methoxy-2-oxoethyl)-2-oxopropyl]-L-alaninamide,or a salt or solvate thereof.

As used herein, the term “zosuquidar” refers to(2R)-1-{4-[(1aR,10bS)-1,1-difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c][7]annulen-6-yl}-3-(quinolin-5-yloxy)propan-2-ol, or a salt or solvate thereof.

Throughout this disclosure, various aspects of the invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range.

For example, description of a range such as from 1 to 6 should beconsidered to have specifically disclosed sub-ranges such as from 1 to3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc.,as well as individual numbers within that range, for example, 1, 2, 2.7,3, 4, 5, 5.1, 5.3, 5.5, and 6. This applies regardless of the breadth ofthe range.

Description

The studies described herein show that a P-gp/BCRP-drivenpharmacoresistance limits the bioavailability of ALS therapeutics usingriluzole, which is the only FDA-approved drug for ALS and a substrate ofP-gp and BCRP. ALS mice (SOD1-G93A) were treated with riluzole andelacridar to block P-gp and BCRP, and monitored for survival as well asbehavioral and physiological parameters. Riluzole, which normally is noteffective when given to a patient at onset of symptoms, was effective inthe ALS mice when administered in combination with the P-gp/BCRPinhibitor elacridar. Chronic elacridar treatment increased riluzole CNSpenetration, improved behavioral measures, including muscle function,slowing down disease progression, and significantly extending survival.The results described herein improved riluzole efficacy with treatmentbeginning at symptom onset.

The impact of P-gp and drug efflux is routinely considered in drugdevelopment, but tests are normally performed in healthy animals orindividuals to determine whether a drug can cross the blood-brainbarrier (BBB). The data presented herein indicates that properpharmacodynamic and pharmacokinetic studies must be analyzed in patientsand disease-relevant models rather than in normal subjects, and thatP-gp and BCRP function, in particular, needs to be considered in thecontext of ALS over disease progression when these two drug effluxtransporters begin to effectively pump out therapeutics and when theirfunction increases incrementally with the disease.

Administration of riluzole along with P-gp blockage effectivelymaintained functional motor neurons, as shown by the CMAPs andbehavioral results, and ultimately slowed disease. Thus, the studiesshow that pharmacologically inhibiting P-gp and BCRP transporteractivity can improve ALS pharmacotherapy.

Riluzole is the only moderately effective drug available for ALStreatment and it is also a substrate for P-gp and BCRP. Additionally,riluzole is the only drug which marginal and inconsistent effects inmice translated into a consistent effect in patients. The data presentedherein clearly demonstrate that, by blocking P-gp and BCRP, it ispossible to enhance riluzole CNS penetration in mice, ultimatelyrestoring its efficacy even when administration begins at onset. Withoutwishing to be limited by any theory, as the data indicate that riluzolepenetration is inversely correlated to riluzole's responsiveness inmice, the decrease in riluzole efficacy observed in patients as diseaseprogresses may derive from the parallel disease-driven increase inpharmacoresistance. As seen in the clinic, there is variability inriluzole efficacy across patients, which may be attributable toindividual variations in expression levels of drug efflux transporters.Revisiting riluzole therapy by inhibiting pharmacoresistance may improvequality of life of ALS patients.

Further, targeting P-gp and BCRP has broader implications for ALStherapeutics that go beyond riluzole itself. P-gp and, to a lesserextent, BCRP have broad substrate specificity, and their activitieslimit bioavailability of multiple drugs. The present data indicate thatthe same mechanism of acquired pharmacoresistance might apply to otherALS therapeutics, and that blocking P-gp and BCRP with elacridar orsimilar transporters' inhibitors might also improve delivery, andultimately, efficacy, of other candidate drugs. In certain embodiments,P-gp and BCRP are the two efflux transporters specifically upregulatedin ALS. In other embodiments, P-gp/BCRP inhibition should be consideredwhen designing future pre-clinical and clinical trials with drugs whichbioavailability is limited by these two transporters. In yet otherembodiments, when designing ALS trials, rather than focusing onincreasing the dose of the drug to maximize its effect, P-gp and BCRPare targeted as an effective way to control and improve bioavailabilityas disease progresses. In yet other embodiments, increasing the ALS drugdose is not be as effective as it might be expected, because diseasepathogenic mechanisms continue to incrementally upregulate P-gp and BCRPexpression levels and function, effectively pumping the drug out of theCNS regardless of the original dose.

Without wishing to be limited by any theory, increasing ALD drug dosesmay not be an effective treatment approach. In one aspect, thesetransporters depend on ATP to extrude their substrates. Thischaracteristic renders them less sensitive to saturation underconditions of ionic unbalances often occurring in disease or insituations of increased substrate concentrations. Regardless of thesubstrate concentrations (e.g., riluzole), ATP-dependent P-gp and BCRPwould still be able to function at no saturation. In another aspect,higher systemic doses of ALS dosing might increase the chances ofhepatotoxicity as the ALS-driven increase in P-gp and BCRP seems to betissue specific, and using elacridar may be the way to just increaseriluzole CNS concentrations without overt toxicity.

Overall, the present studies suggest that to maintain efficacious levelsof therapeutics throughout disease progression adjusting doses ofelacridar (or other more potent and selective P-gp/BCRP inhibitors) maybe the most valuable solution. This approach may be used to improve theeffects of riluzole in patients as well as reevaluate other drugs thatfailed in preclinical and clinical trials. In certain embodiments,evaluation of the contribution of P-gp and BCRP altering drugbioavailability and therapeutic efficacy of new ALS therapeutics isperformed on a drug-to-drug and patient-to-patient basis.

Compositions

The compounds included in the compositions useful within the inventionmay be obtained from commercial sources and/or synthesized usingtechniques well-known in the art of organic synthesis.

The compositions useful within the invention may include aneurodegenerative disease drug or a salt or solvate thereof. In certainembodiments, the neurodegenerative disease comprises ALS. In otherembodiments, the drug is a substrate of an ABC transporter inhibitor. Inyet other embodiments, the ABC transporter comprises at least oneselected from the group consisting of P-gp and BRCP.

In certain embodiments, the neurodegenerative disease comprises at leastone selected from the group consisting of spinal cord injury,Alzheimer's disease, Parkinson's disease, Huntington's disease, priondisease, amyotrophic lateral sclerosis, a tauopathy, and chronictraumatic encephalopathy.

In certain embodiments, the neurodegenerative drug comprises at leastone selected from the group consisting of: ceftriaxone (also known as(6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino)acetyl]amino}-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylicacid); celecoxib (also known as4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide);ciliary neurotrophic factor; cobalamin (including cyano-, hydroxo-,methyl-, and adenosyl-cobalamain); coenzyme Q (also known as2-[(2E,6E,10E,14E,18E,22E,26E,30E,34E)-3,7,11,15,19,23,27,31,35,39-decamethyltetraconta-2,6,10,14,18,22,26,30,34,38-decaenyl]-5,6-dimethoxy-3-methylcyclohexa-2,5-diene-1,4-dione);gabapentin (also known as 2-[1-(aminomethyl)cyclohexyl]acetic acid); HGF(hepatocyte growth factor); IGF-I (insulin-like growth factor 1);minocycline (also known as(2E,4S,4aR,5aS,12aR)-2-(amino-hydroxy-methylidene)-4,7-bis(dimethylamino)-10,11,12a-trihydroxy-4a,5,5a,6-tetrahydro-4H-tetracene-1,3,12-trione);N-acetylcysteine; NDGA (also known as nordihydroguaiaretic acid, or4,4′-(2,3-dimethylbutane-1,4-diyl)dibenzene-1,2-diol); pentoxifylline(also known as3,7-dimethyl-1-(5-oxohexyl)-3,7-dihydro-1H-purine-2,6-dione); riluzole(also known as 6-(trifluoromethoxy) benzothiazol-2-amine); thalidomide(also known as(RS)-2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione); topiramate(also known as 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranosesulfamate); valproic acid (also known as 2-propylpentanoic acid); VEGF(vascular endothelial growth factor); vitamin E; zVAD-fmk (also known asbenzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone); a salt orsolvate thereof, and mixtures thereof.

The compositions useful within the invention may include an ABCtransporter inhibitor. In certain embodiments, the ABC transportercomprises at least one selected from the group consisting of P-gp andBRCP.

In certain embodiments, the ABC transporter inhibitor includes at leastone selected from the group consisting of: elacridar (also known asN-[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide);tariquidar (also known asN-[2-[[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide);zosuquidar (also known as(2R)-1-{4-[(1aR,10bS)-1,1-difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa [c][7]annulen-6-yl}-3-(quinolin-5-yloxy)propan-2-ol);ONT-093 (also known as(E)-4,4′-(2-(4-(3-ethoxyprop-1-en-1-yl)phenyl)-1H-imidazole-4,5-diyl)bis(N-isopropylaniline));laniquidar (also known as methyl11-(1-{2-[4-(quinolin-2-ylmethoxy)phenyl]ethyl}piperidin-4-ylidene)-6,11-dihydro-5H-imidazo[2,1-b][3]benzazepine-3-carboxylate);a salt or solvate thereof, and any mixtures thereof.

The compositions useful within the invention may include apharmaceutically acceptable carrier. In certain embodiments, theneurodegenerative drug and the ABC transporter inhibitor areco-formulated.

Methods

The invention includes a method of treating a neurodegenerative diseasein a mammal in need thereof. The method comprises administering to themammal a therapeutically effective amount of a neurodegenerative diseasedrug, wherein the drug is a substrate of an ABC transporter. Accordingto the method, the mammal is further administered a therapeuticallyeffective amount of an ABC transporter inhibitor, whereby theneurodegenerative disease is treated in the mammal.

In certain embodiments, the neurodegenerative disease comprises at leastone selected from the group consisting of spinal cord injury,Alzheimer's disease, Parkinson's disease, Huntington's disease, priondisease, ALS, a tauopathy, and chronic traumatic encephalopathy. Inother embodiments, the neurodegenerative disease comprises ALS. In yetother embodiments, the ABC transporter comprises at least one selectedfrom the group consisting of P-gp and BRCP.

In certain embodiments, the drug comprises at least one selected fromthe group consisting of ceftriaxone; celecoxib; ciliary neurotrophicfactor; cobalamin; coenzyme Q; gabapentin; HGF; IGF-I; minocycline;N-acetylcysteine; NDGA; pentoxifylline; riluzole; thalidomide;topiramate; valproic acid; VEGF; vitamin E; zVAD-fmk; a salt or solvatethereof, and mixtures thereof. In other embodiments, the ABC transporterinhibitor comprises at least one selected from the group consisting ofelacridar; tariquidar; zosuquidar; ONT-093; laniquidar; a salt orsolvate thereof, and mixtures thereof.

In certain embodiments, the drug is part of a pharmaceuticalcomposition. In other embodiments, the inhibitor is part of apharmaceutical composition. In other embodiments, the pharmaceuticalcomposition comprises an extended-release formulation.

In certain embodiments, the drug is administered to the mammal beforethe inhibitor. In other embodiments, the inhibitor is administered tothe mammal before the drug. In yet other embodiments, the inhibitor andthe drug are administered to the mammal at about the same time. In yetother embodiments, the drug and the inhibitor are co-administered to themammal. In yet other embodiments, the drug and the inhibitor arecoformulated. In yet other embodiments, the drug and/or the inhibitoris/are administered at or after the onset of any symptom of the disease.

In certain embodiments, the mammal that is administered the drug and theinhibitor has a higher spinal cord concentration of the drug than amammal that is administered the drug only. In certain embodiments, themammal that is administered the drug and the inhibitor has a highercompound muscle action potential peak amplitude than a mammal that isadministered the drug only. In certain embodiments, the mammal that isadministered the drug and the inhibitor has improved survival ascompared to a mammal that is administered the drug only. In certainembodiments, the mammal that is administered the drug and the inhibitorhas delayed disease progression as compared to a mammal that isadministered the drug only.

In certain embodiments, the mammal is a rodent or a primate. In otherembodiments, the primate is a human. In yet other embodiments, the drugis administered to the mammal by at least one route selected from thegroup consisting of inhalational, oral, rectal, vaginal, parenteral,topical, transdermal, pulmonary, intranasal, buccal, sublingual,ophthalmic, intrathecal, intravenous and intragastrical.

Combination Therapies

The compositions useful within the present invention are intended to beuseful in the methods of present invention in combination with one ormore additional compounds useful for treating the diseases or disorderscontemplated within the invention. These additional compounds maycomprise compounds of the present invention or compounds, e.g.,commercially available compounds, known to treat, prevent, or reduce thesymptoms of the diseases or disorders contemplated within the invention.

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 1981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22: 27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the patienteither prior to, around the time, or after the onset of a disease ordisorder. Further, several divided dosages, as well as staggered dosagesmay be administered daily or sequentially, or the dose may becontinuously infused, or may be a bolus injection. Further, the dosagesof the therapeutic formulations may be proportionally increased ordecreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the compositions useful within the present inventionto a patient, such as a mammal, such as a human, may be carried outusing known procedures, at dosages and for periods of time effective totreat a disease or disorder in the patient. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the state of the disease or disorder in thepatient; the age, sex, and weight of the patient; and the ability of thetherapeutic compound to treat a disease or disorder in the patient.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. For example, several divided doses may be administered dailyor the dose may be proportionally reduced as indicated by the exigenciesof the therapeutic situation. In certain embodiments, an effective doserange for a therapeutic compound of the invention ranges from about 1 toabout 5,000 mg/kg of body weight/day. In other embodiments, an effectivedose range for a therapeutic compound of the invention ranges from about100 to about 1,000 mg/kg of body weight/day. In yet other embodiments,an effective dose range for a therapeutic compound of the inventionranges from about 10 to about 50 mg/kg of body weight/day. In yet otherembodiments, an effective dose range for a therapeutic compound of theinvention ranges from about 1 to about 50 mg/kg of body weight/day. Inyet other embodiments, an effective dose range for a therapeuticcompound of the invention ranges from about 1 to about 10 mg/kg of bodyweight/day. In yet other embodiments, an effective dose range for atherapeutic compound of the invention is about 2.5 mg/kg of bodyweight/day. One of ordinary skill in the art would be able to study therelevant factors and make the determination regarding the effectiveamount of the therapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease or disorder in a patient.

In certain embodiments, the compositions useful within the invention areformulated using one or more pharmaceutically acceptable excipients orcarriers. In certain embodiments, the pharmaceutical compositions of theinvention comprise a therapeutically effective amount of a compounduseful within the invention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it may include isotonic agents, for example, sugars, sodiumchloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

In certain embodiments, the compositions useful within the invention areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions useful withinthe invention are administered to the patient in range of dosages thatinclude, but are not limited to, once every day, every two, days, everythree days to once a week, and once every two weeks. It will be readilyapparent to one skilled in the art that the frequency of administrationof the various combination compositions useful within the invention willvary from individual to individual depending on many factors including,but not limited to, age, disease or disorder to be treated, gender,overall health, and other factors. Thus, the invention should not beconstrued to be limited to any particular dosage regime and the precisedosage and composition to be administered to any patient will bedetermined by the attending physical taking all other factors about thepatient into account.

Compounds for administration may be in the range of from about 1 μg toabout 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg,about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg toabout 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg,about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mgto about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500mg, and any and all whole or partial increments therebetween.

In certain embodiments, the dose of a compound is from about 1 mg andabout 2,500 mg. In other embodiments, a dose of a compound of theinvention used in compositions described herein is less than about10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, orless than about 5,000 mg, or less than about 3,000 mg, or less thanabout 2,000 mg, or less than about 1,000 mg, or less than about 500 mg,or less than about 200 mg, or less than about 50 mg. Similarly, in otherembodiments, a dose of a second compound (i.e., a drug used for treatinga disease or disorder) as described herein is less than about 1,000 mg,or less than about 800 mg, or less than about 600 mg, or less than about500 mg, or less than about 400 mg, or less than about 300 mg, or lessthan about 200 mg, or less than about 100 mg, or less than about 50 mg,or less than about 40 mg, or less than about 30 mg, or less than about25 mg, or less than about 20 mg, or less than about 15 mg, or less thanabout 10 mg, or less than about 5 mg, or less than about 2 mg, or lessthan about 1 mg, or less than about 0.5 mg, and any and all whole orpartial increments thereof.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof a disease or disorder in a patient.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other cognition improving agents.

The term “container” includes any receptacle for holding thepharmaceutical composition. For example, in certain embodiments, thecontainer is the packaging that contains the pharmaceutical composition.In other embodiments, the container is not the packaging that containsthe pharmaceutical composition, i.e., the container is a receptacle,such as a box or vial that contains the packaged pharmaceuticalcomposition or unpackaged pharmaceutical composition and theinstructions for use of the pharmaceutical composition. Moreover,packaging techniques are well known in the art. It should be understoodthat the instructions for use of the pharmaceutical composition may becontained on the packaging containing the pharmaceutical composition,and as such the instructions form an increased functional relationshipto the packaged product. However, it should be understood that theinstructions may contain information pertaining to the compound'sability to perform its intended function, e.g., treating, preventing, orreducing a disease or disorder in a patient.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients which are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, the compounds may be in the form of tablets orcapsules prepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., polyvinylpyrrolidone,hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g.,cornstarch, lactose, microcrystalline cellulose or calcium phosphate);lubricants (e.g., magnesium stearate, talc, or silica); disintegrates(e.g., sodium starch glycollate); or wetting agents (e.g., sodium laurylsulphate). If desired, the tablets may be coated using suitable methodsand coating materials such as OPADRY™ film coating systems availablefrom Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, OrganicEnteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™White, 32K18400). Liquid preparation for oral administration may be inthe form of solutions, syrups or suspensions. The liquid preparationsmay be prepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) will melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of a disease or disorder. Using awax/pH-sensitive polymer mix, a gastric insoluble composition may beobtained in which the active ingredient is entrapped, ensuring itsdelayed release.

Parenteral Administration

For parenteral administration, the compounds may be formulated forinjection or infusion, for example, intravenous, intramuscular orsubcutaneous injection or infusion, or for administration in a bolusdose and/or continuous infusion. Solutions, suspensions or emulsions inan oily or aqueous vehicle, optionally containing other formulatoryagents such as suspending, stabilizing and/or dispersing agents may beused.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389,5,582,837, and 5,007,790 Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.2003/0147952, 2003/0104062, 2003/0104053, 2003/0044466, 2003/0039688,and 2002/0051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041, WO03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In certain embodiments of the invention, the compounds of the inventionare administered to a patient, alone or in combination with anotherpharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that may,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound will dependon the age, sex and weight of the patient, the current medical conditionof the patient and the progression of the disease or disorder in thepatient being treated. The skilled artisan will be able to determineappropriate dosages depending on these and other factors.

A suitable dose of a compound of the present invention may be in therange of from about 0.01 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

The compounds for use in the method of the invention may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials and Methods: Animals:

Mice were housed in accordance with Thomas Jefferson UniversityInstitutional Animal Care and Use Committee (IACUC) and the NIH Guidefor the Care and Use of Laboratory Animals. Mutant SOD1-G93A micemodeling ALS [B6.Cg-Tg(SOD1-G93A)1Gur/J] were purchased from JacksonLaboratories (Sacramento, Calif., catalog no. 004435). Male SOD1-G93Amice were bred with C57BL/6 females. Offspring were genotyped todetermine presence of the human SOD1 transgene. As transgene copy numberis known to fluctuate and decrease through subsequent breedinggenerations, quantitative RT-PCR was performed to exclude mice withdecreased SOD1 human transgene copy number. P-glycoprotein knockout mice(P-gp^(−/−)) on the FVB/N background were purchased from Taconic(Germantown, N.Y., catalog no. #1487). Prior to crossing with SOD1-G93Amice, P-gp^(−/−) mice were backcrossed to C57BL/6 mice (N=5). After thefifth generation and a homogeneous B6SJL background, P-gp^(−/−) micewere crossed with SOD1-G93A mice and genotyped to determine presence ofthe SOD1-G93A human transgene. Mice were controlled for human transgenecopy number via qRT-PCR.

Drug Treatment and Survival Analysis:

Transgenic mutant SOD1-G93A male mice were used in a preclinical drugstudy format. Mice were enrolled into one of three treatment groups:control+placebo/elacridar (n=25), riluzole+placebo (n=25), orriluzole+elacridar (n=25). Riluzole was administered via the chow (125mg/kg of chow). Elacridar was administered via a time-controlled releasepellet (50 mg/10 day release, Innovative Research of America, Sarasota,Fla.), which was implanted subcutaneously in the back of the neck.Control chow was prepared in the same manner as the riluzole chow. Tocontrol for the effects of surgical pellet implantation, control andriluzole-treated mice also received placebo pellets every 10 days.

Treatment of riluzole and elacridar began at 100 days of age andcontinued for the lifespan of all mice. Baseline and study measures wererecorded for weight, grip strength, food consumption, and survival.Determination of end-stage was the inability for a mouse to right itselfafter 30 seconds of being placed on its side.

Grip Strength and Weight Assessment:

Hindlimb grip strength was recorded 2-3times per week for each mouse.The rate of grip strength deterioration for riluzole+placebo andriluzole+elacridar groups was determined based on the average gripstrength per day per mouse and fit to a linear curve. The weights foreach mouse was recorded 2-3times per week for each mouse and the rate ofdecline was quantified for riluzole+placebo and riluzole+elacridargroups.

Compound Muscle Action Potential (CMAP) Recordings:

Mice were anesthetized with 1% isoflurane and 0.5% oxygen. Followingdermatomoty, stainless steel-timulating needle electrodes were insertedat the sciatic notch, near the sciatic nerve. A ground electrode wasplaced subcutaneously in the back, and a reference electrode placedsubcutaneously at the ankle. The sciatic nerve was stimulated (0.2 msduration; 1.6 mV amplitude) and the response recorded via a needleelectrode inserted into the plantar muscle in the medial half of thefoot, following the line connecting the first and fifthtarsal/metatarsal joints. Data was collected using ADI Powerlab 8/30stimulator and BioAMP amplifier (ADInstruments, Colorado Springs, Colo.)and analyzed using Scope 3.5.6 (ADInstruments). The compound muscleaction potential (M-wave) amplitude was measured from baseline to peak.Data was averaged between left and right hind limb traces.

In Vivo LD800 Imaging:

LD800 (1 mg/kg) was injected i.p. Twenty minutes later animals wereanesthetized with a ketamine:xylazine mixture and sacrificed via cardiacperfusion with heparinized PBS. Spinal cord samples were collected andembedded in low-melting point agarose and sectioned in 750 μm thicksections using a tissue chopper (McIlwain Tissue Chopper, The MickleLaboratory Engineering Company). Spinal cord tissue sections were thenimaged with an Odyssey infrared imager (LI-COR Biosciences).

Mass Spectrometry:

Age-matched, adult mice (150 days of age) were injectedintraperitoneally with Riluzole.HCl (12 mg/kg). After 1 h, mice weresacrificed with carbon dioxide, blood was collected via cardiacpuncture, and mice were flushed with saline (0.9%). Blood was placed inEDTA-coated eppendorf tubes and centrifuged to collect plasma. Brainsand spinal cords were harvested and flash frozen. All samples werestored at −80° C. until extraction. For plasma samples, nine volumesice-cold, 100% ethanol were added to each sample, incubated overnight at4° C. and centrifuged for 25 min (3,500 rpm, 4° C.). Ethanol supernatantwas removed and pellets were dried in a speed vacuum. Pellets werestored at −80° C. Pellets were resuspended in 0.1% formic acid, waterbath sonicated for 20 min, and centrifuged at 15 rcf for 30 min.Supernatant was further diluted in 0.1% formic acid, and analyzed byLC-MS/MS on a Thermo LTQ-Orbitrap XL mass spectrometer at the WistarProteomics Facility. Riluzole had elution peaks at 17 min, peak areaswere used at 5 ppm Mass Accuracy to find relative concentration amountsfrom standard curve.

Motor Neuron Counts:

Mice were perfused with Dulbecco's phosphate-buffered saline (DPBS) andspinal cord lumbar segments were embedded in OCT freezing medium andstored at −20° C. until sectioned. Forty micrometer fresh frozen tissuesections were dissected using a temperature controlled cryostat (MICROMHM 505 E). Tissue sections were coded blind and processedsimultaneously. Sections were fixed with 2.5% paraformaldehyde for 10min at room temperature. Every second section was stained about 3-4 minwith 0.1% cresyl violet acetate and dipped in 95% Ethanol and 95%ethanol+glacial acetic acid to facilitate the staining. Finally, slideswere mounted with permount and dried overnight at room temperature.Stained sections were visualized under a 60× oil immersion objective ofa bright-field microscope (Olympus). Four sections per animal wereanalyzed per group (n=7). Large pyramidal motor neurons positivelystained for cresyl violet with a prominent nucleus and size at least ≧20μm² were counted. Motor neurons were quantified using the opticalfractionator workflow module of stereo investigator software (version 8)employing a grid size 75×75 μm and sampling grid size 100×100 μm. Allanalysis was carried out by an investigator blinded to the samples.Motor neuron counts per group were later quantified using Student'st-test and represented as average±SEM.

Periodic Acid Schiff Staining:

Saline perfused liver was postfixed with 4% paraformaldehyde for 24 hfollowed by 24 h incubation in 30% sucrose solution, embedded in OCTsolution, and stored at −80° C. until sectioned (10 μm thickness).Sections were hydrated with water, immersed in PAS solutions (Sigma;395) for 5 min at room temperature, and washed in water. Sections wereimmersed for 4 min in Schiff's reagent and washed in water for 5 min.Sections were counterstained with hematoxylin solution, dehydrated inethanol and HemoD solution, mounted, and imaged.

Immunofluorescence of Patient Tissue:

OCT-embedded spinal cord and hippocampus tissue were cryostat sectionedat 10 μm. Slides were rinsed once in 1.5× TBS buffer and postfixed in 4%paraformaldehyde for 10 min at room temperature followed by treatmentwith antigen unmasking solution for 2 min at −20° C. (33% acetic acidand 66% ethanol). After washing and blocking (2% BSA, 0.3% triton-X, 5%horse serum in 1.5× TBS), slides were incubated with primary antibodies(C219 from Covance 1:50 and vWF from Dako 1:50). After washing, tissuewas then incubated with fluorescent secondary antibodies, mounted withDAPI antifade solution, and imaged.

Example

Homogenates of lumbar spinal cords of two sporadic and two familial ALSpatients displayed increased levels of P-gp compared to controls,including a control patient with Friedreich's Ataxia (FIG. 1A). Byimmunohistochemistry, increases in P-gp expression were found inendothelial cells of the BSCB (FIGS. 1B-1C). A selective,tissue-specific increase of P-gp expression was found in areas affectedby the disease, specifically spinal cord, while P-gp levels remained lowin the unaffected hippocampus (FIG. 1C). Microvascular leakage andreduced tight junction protein expression are known to occur in ALS andcould contribute to motor neuron damage. Without wishing to be limitedby any theory, upregulation of P-gp may occur as a compensatory responseto a leaking BSCB.

In certain embodiments, activation of P-gp can prevent effective drugdelivery in the CNS, reducing drug bioavailability. To demonstrate this,the in vivo accumulation of a specific P-gp substrate, LD800, wasexamined in the spinal cord of the ALS mouse model where expression andfunction of P-gp increases as disease progresses. Symptomatic mice had amarked decrease in LD800 accumulation as compared to nontransgenic andP-gp^(−/−) mice (FIGS. 2A-2B).

The extent to which P-gp alters riluzole disposition into the CNS wasanalyzed. The percent of riluzole accumulation in the spinal cord andbrain was higher in P-gp^(−/−) mice compared to age-matched,non-transgenic mice (FIGS. 2C-2D). Then, P-gp^(−/−) and SOD1-G93A micewere crossed to obtain ALS mice lacking P-gp(P-gp^(−/−)::SOD1-G93A^(+/−)). These mice were treated with riluzolebeginning at symptom onset (at 100 days of age). As very low numbers ofP-gp^(−/−)::SOD1-G93A mice were obtained from these crosses, theanalysis was limited to 5-6 mice per group to allow initial evaluationof the impact of P-gp knock down on disease and riluzole penetration.Knocking out P-gp from the ALS mice improved significantly thetherapeutic effect of riluzole (FIG. 2E), further strengthening theassumption that as ALS progresses in mice and P-gp and BCRP increase,riluzole penetration and efficacy decrease.

To compensate for this progressive decrease in therapeutic levels ofriluzole in the spinal cord of diseased mice, the activity of P-gp andBCRP was pharmacologically inhibited. Inhibitors of drug effluxtransporters can be used to restore drug sensitivity in diseases inwhich the issue of pharmacoresistance is well characterized, such asleukemia, other forms of cancer, and epilepsy. Third-generationinhibitors, such as elacridar (GF120918), are highly specific andtolerated in patients. The mice were treated with elacridar, whichprovides dual inhibition of P-gp and BCRP. Chronic elacridar treatment,beginning at disease onset, was safe and did not alter diseaseprogression in SOD1-G93A mice (FIG. 3A). As expected, chronic treatmentof elacridar did not affect overall expression of P-gp (FIG. 3B) orBCRP; it did inhibit their function, though, resulting in a significantincrease in riluzole spinal cord concentrations in diseased mice (FIG.3C). Despite higher levels of systemic riluzole, theriluzole+elacridar-treated mice did not show signs of liver pathology(FIG. 3D), indicating that elacridar did not cause liver toxicity andthat increasing systemic riluzole concentrations via elacridar were nothepatotoxic.

To determine whether the increased drug bioavailability translated intoimproved efficacy, riluzole was tested as the candidate drug. This drughas a marginal, albeit variable, effect in the SOD1-G93A mice. Theriluzole and elacridar treatment was initiated in the mouse models. Mostpublished preclinical studies in mice began riluzole treatmentpresymptomatically (between 50 and 60 days) and prior to disease onset.This is based on the assumption that high levels of mutant SOD1expression in mice lead to an “enhanced” disease that needs to bemanaged aggressively. But most of the compounds testedpresymptomatically have only delayed disease onset rather than sloweddisease progression with an overall extension of lifespan <10%. Withoutwishing to be limited by any theory, a delay in onset withpharmacological treatment is not likely to be predictive of how well adrug may perform clinically.

The prior observations showed that P-gp and BCRP function increasedbeginning at symptom onset and peaked at symptomatic stage, so theriluzole and elacridar treatment was initiated at onset (P100). Inaddition, without wishing to be limited by any theory, the study canmimic, to the extent possible, the therapeutic regimen of the patientswho are treated with riluzole after they are diagnosed with the disease.

The analysis of the SOD1-G93A mice consisted of three groups of mice(n=25 males/group): (1) control, placebo-treated; (2)riluzole+placebo-treated; and (3) riluzole+elacridar-treated. Copynumber of the SOD1-G93A human transgene was controlled (FIG. 3E).Cotreatment using riluzole+elacridar beginning at symptoms onsetsignificantly extended survival compared to control/placebo andriluzole/placebo groups (FIG. 4A). Riluzole alone was not beneficial anddid not extend survival compared to control mice. However, when given incombination with elacridar, it significantly slowed disease progression,extending mouse survival by 13% from onset of symptoms (FIG. 4A). Thisspecific effect on disease progression was superior to manypharmacological interventions in ALS mice, which primarily affecteddisease onset but not duration.

Consecutive surgeries of pellet implantation had negative effects on themice. The life-span of placebo pellet-implanted SOD1-G93A mice decreasedby ˜6 days compared to nonsurgery ALS mice (156.7±2.8 days and162.9±0.69 days, respectively; −3.8%; P<0.05), likely due to thenegative impact of multiple pellet implantation surgeries. In certainembodiments, the full potential of the beneficial effect ofriluzole+elacridar might have been negatively skewed by the pelletimplantation regimen.

Treatment of riluzole and elacridar significantly improved vitalparameters (FIG. 4). Two additional cohorts of mice were treated withriluzole+placebo (n=10) or riluzole+elacridar (n=11), from 100 to 140days, and electrophysiological recordings from the plantar musclefollowing sciatic nerve stimulation were performed to measure functionalinnervation by motor neurons of the lumbar spinal cord (FIGS. 4B-4C).CMAPs had higher peak amplitudes in 140 day riluzole/elacridar micecompared to age-matched riluzole/placebo mice (FIGS. 4B-4C). Throughoutthe 40-day treatment, the riluzole/elacridar mice maintainedsignificantly higher CMAP values compared to the riluzole/placebo mice(FIG. 4C). In the same mice, hindlimb grip strength was improved (FIG.4E). There was a trend for an increased number of remaining motorneurons in the lumbar spinal cord of P140 mice cotreated with riluzoleand elacridar. In certain embodiments, stopping motor neuron death maynot be sufficient to slow disease. In other embodiments, maintainingmotor neuron function may be sufficient to slow disease. In yet otherembodiments, there is a dissociation between motor neuron death anddisease progression in mutant SOD1 mice.

In a non-limiting aspect, accounting for the contribution of ABCtransporters to ALS pharmacoresistance may both improve the modesteffects of riluzole therapy and allow for a reevaluation of previouslydiscarded ALS drugs.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of treating or ameliorating aneurodegenerative disease in a mammal in need thereof, the methodcomprising administering to the mammal a therapeutically effectiveamount of a neurodegenerative disease drug, wherein the drug is asubstrate of an ABC transporter, wherein the mammal is furtheradministered a therapeutically effective amount of an ABC transporterinhibitor, whereby the neurodegenerative disease is treated orameliorated in the mammal.
 2. The method of claim 1, wherein theneurodegenerative disease comprises at least one selected from the groupconsisting of spinal cord injury, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, prion disease, amyotrophic lateralsclerosis, a tauopathy, and chronic traumatic encephalopathy. 3.(canceled)
 4. The method of claim 1, wherein the ABC transportercomprises at least one selected from the group consisting of P-gp andBRCP.
 5. The method of claim 1, wherein the drug comprises at least oneselected from the group consisting of ceftriaxone; celecoxib; ciliaryneurotrophic factor; cobalamin; coenzyme Q; gabapentin; HGF; IGF-I;minocycline; N-acetylcysteine; NDGA; pentoxifylline; riluzole;thalidomide; topiramate; valproic acid; VEGF; vitamin E; zVAD-fmk; asalt or solvate thereof, and mixtures thereof.
 6. The method of claim 1,wherein the inhibitor comprises at least one selected from the groupconsisting of elacridar; tariquidar; zosuquidar; ONT-093; laniquidar; asalt or solvate thereof, and mixtures thereof.
 7. (canceled) 8.(canceled)
 9. The method of claim 1, wherein the drug and the inhibitorare co-administered to the mammal.
 10. The method of claim 9, whereinthe drug and the inhibitor are coformulated.
 11. The method of claim 1,wherein administration of the drug and the inhibitor to the mammal takesplace at the time or after the mammal develops at least one symptom ofthe neurodegenerative disease.
 12. The method of claim 1, wherein themammal that is administered the drug and the inhibitor has a higherspinal cord concentration of the drug than a mammal that is administeredthe drug only.
 13. The method of claim 1, wherein the mammal that isadministered the drug and the inhibitor has a higher compound muscleaction potential peak amplitude than a mammal that is administered thedrug only.
 14. The method of claim 1, wherein the mammal that isadministered the drug and the inhibitor has improved survival ascompared to a mammal that is administered the drug only.
 15. The methodof claim 1, wherein the mammal that is administered the drug and theinhibitor has delayed disease progression as compared to a mammal thatis administered the drug only.
 16. (canceled)
 17. (canceled)
 18. Themethod of claim 1, wherein the drug is administered to the mammal by atleast one route selected from the group consisting of inhalational,oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary,intranasal, buccal, sublingual, ophthalmic, intrathecal, intravenous andintragastrical.
 19. A pharmaceutical composition comprising aneurodegenerative disease drug and an ABC transporter inhibitor, whereinthe drug is a substrate of an ABC transporter.
 20. The composition ofclaim 19, wherein the disease comprises at least one selected from thegroup consisting of spinal cord injury, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, prion disease, amyotrophic lateralsclerosis, a tauopathy, and chronic traumatic encephalopathy.
 21. Thecomposition of claim 19, wherein the disease comprises amyotrophiclateral sclerosis.
 22. The composition of claim 19, wherein the ABCtransporter comprises at least one selected from the group consisting ofP-gp and BRCP.
 23. The composition of claim 19, wherein the drugcomprises at least one selected from the group consisting ofceftriaxone; celecoxib; ciliary neurotrophic factor; cobalamin; coenzymeQ; gabapentin; HGF; IGF-I; minocycline; N-acetylcysteine; NDGA;pentoxifylline; riluzole; thalidomide; topiramate; valproic acid; VEGF;vitamin E; zVAD-fmk; a salt or solvate thereof, and mixtures thereof.24. The composition of claim 19, wherein the inhibitor comprises atleast one selected from the group consisting of elacridar; tariquidar;zosuquidar; ONT-093; laniquidar; a salt or solvate thereof, and mixturesthereof.
 25. The composition of claim 19, wherein the composition isformulated for inhalational, oral, rectal, vaginal, parenteral, topical,transdermal, pulmonary, intranasal, buccal, sublingual, ophthalmic,intrathecal, intravenous or intragastrical administration.